The overall objective of this proposal is to define the mechanisms involved in allograft acceptance with particular emphasis on the analysis of the qualitative and quantitative changes on the regulatory cell populations which influences the immune system towards graft adaptation. Using a newly developed sensitive and reproducible limiting dilution analysis (LDA) technique, we have recently obtained evidence for specific clonal reduction, decreased helper T cell precursor frequency and the presence of specific regulatory cell(s) which suppresses host anti-donor cell-mediated cytotoxicity in longterm human leukocyte antigen (HLA) disparate renal allograft recipients. The proposed research will determine quantitatively the cytolytic T cell precursor (CTLp), helper T cell precursor (HTLp), and the immunoregulatory cell precursor (IRp) frequencies in longterm, HLA disparate, living-related transplant recipients by LDA. In order to define the influence of various major histocompatibility complex (MHC) disparities on the frequency of CTLp, HTLp and IRp, peripheral blood mononuclear cells from healthy humans will be stimulated in LDA with allogeneic cells expressing various combinations of HLA class I and/or class II alloantigens. This will also enable us to identify the relative importance of various HLA antigens quantitatively so that appropriate MHC matching can be rationalized for future transplants. It is also proposed to analyze serially transplant recipients who receive donor specific blood transfusion (DST) in order to understand the influence of DST on the number of alloantigen reactive CTLp, HTLp, and IRp and their correlation with episodes of rejection. Finally, the phenotypic and functional characterization of the immunoregulatory cells will be determined using monoclonal antibodies specific for CD4, CD8, and/or CD28 antigens, panning and isolation of subpopulation, and idiotype specific cellular adsorption techniques using mixed lymphocyte culture stimulated syngeneic blast. Attempts will also be made to propagate and clone In vitro the active regulatory cells using recently reported culture conditions.